During cerebral cortical development, after closure of the anterior neural tube, neuroepithelial cells proliferate within the walls of the ventricle, populating the ventricular zone (VZ) with a pseudostratified layer of bipolar cells (). At the onset of neurogenesis, neuroepithelial cells transform into radial glial progenitors (RGPs), which are characterized by a highly polarized morphology and a glial-like molecular identity (reviewed in Götz and Huttner 2005
). Initially, RGPs expand their population by dividing symmetrically to produce 2 daughter RGPs. Then, as neurogenesis proceeds, an increasing number of RGPs begin to divide asymmetrically, giving rise to a daughter neuron or intermediate progenitor (IP) and a self-renewing daughter RGP (Malatesta et al. 2000
; Miyata et al. 2001
; Anthony et al. 2004
; Noctor et al. 2004
). IPs move to the subventricular zone (SVZ), where they form an additional neurogenic niche that supplies the majority of projection neurons to all layers of the cerebral cortex (Haubensak et al. 2004
; Kowalczyk et al. 2009
). As neurogenesis wanes, asymmetric neurogenic RGP divisions decrease in frequency due to the terminal differentiation of RGPs into glia and ependymal cells (Noctor et al. 2008
) (). During neurogenesis, significant molecular and morphological heterogeneity among neural progenitors in the VZ has been characterized and this heterogeneity may be essential to create neuronal diversity in cerebral cortex (Gal et al. 2006
; Mizutani et al. 2007
; Anthony and Heintz 2008
; Howard et al. 2008
; Kawaguchi et al. 2008
; Hansen et al. 2010
; Stancik et al. 2010
Figure 1. Progression of radial progenitor development in embryonic cerebral cortex. Initially, RGPs are established from an undifferentiated sheet of neuroepithelial cells (A). Radial glia then divide symmetrically and generate more radial glia (B). Subsequent (more ...)
In addition to their role as cortical progenitors, RGPs perform another important function as guides for neuron migration. Specifically, the pial-directed radial process of RGPs provides a permissive and instructive scaffolding for the oriented migration and placement of newly generated neurons (Ayala et al. 2007
). Thus, as neuronal progenitors and migratory guides, RGPs perform multiple evolving functions as cortical development unfolds (), making it necessary to develop tools to analyze each of these functions.
Time-lapse Analysis of Adherent Neural Progenitor Clones
A fundamental question in radial progenitor development during corticogenesis is what governs the timing and mode of RGP division and the gradual restriction of daughter cell fate as development proceeds. Is the sequential production of RGPs, IPs and neurons, and then glia regulated by cell-intrinsic programs, extrinsic cues, or a combination of both? Similarly, what signals impinging on progenitors determine the orderly generation of layer-specific projection neurons?
In vitro assays using isolated, single cortical progenitors from different embryonic ages can be used to selectively study and manipulate RGP proliferation, differentiation, and cell fate in a defined environment. In this method, dissociated cortical progenitors are plated on an adherent substrate at clonal density, so that cell–cell contacts are minimized, and the only extracellular cues present are produced from the clones themselves or exogenously added to the defined culture medium (Shen et al. 2006
). Over several days in vitro, single progenitors generate other progenitors, distinct neuronal subtypes, and glia, with a timing and order that parallels the timing and order observed in vivo (Shen et al. 2006
). The identity of daughter cells generated from isolated progenitors can be determined using cell type–specific markers, thus defining the mode of progenitor division. For instance, if an isolated progenitor gives rise to only RGPs, it underwent symmetric self-renewing divisions (). If the daughter cells are of different cell types (i.e., neurons, intermediate precursor, or astroglia), they are likely the result of asymmetric divisions (). While this assay is ideal to test the cell-intrinsic capacities of cortical progenitors from different developmental stages (), extrinsic cues critical for distinct patterns of progenitor division can also be presented to the isolated clones and tested for their influence on the mode of progenitor division.
Time-lapse analysis of neural progenitor clones is a further extension of this technique where long-term live-cell imaging of isolated progenitors and their descendents (), followed by correlative staining for progenitor, neuronal, and glial subtypes, can be used to establish a comprehensive lineage tree (Al-Kofahi et al. 2006
; Shen et al. 2006
). The effects of extrinsic factors or cell–cell contacts on progenitor division can be examined by adding diffusible cues to the medium or culturing progenitors at higher density, respectively (Ravin et al. 2008
; Shen et al. 2006
). Additional refinements to the system might include methods to specify the identity of the initial progenitors. For example, one can use fluorescence activated cell sorting to purify progenitors from transgenic mice expressing X
FP under the control of a promoter active in a specific progenitor subpopulation (e.g., BLBP-GFP to isolate RGPs, Tα1-GFP to isolate short neural precursors, or Tbr2-GFP to isolate IPs; ).
Figure 2. Analysis of isolated cortical progenitor development. Radial progenitors micro dissected from the VZ of E14.5 BLBP-GFP embryonic cortices were plated at clonal density and isolated, and genetically defined radial progenitors (GFP+) were time-lapse imaged. (more ...)
Progenitor-specific transgenic mouse lines
The advantage of this technique over neurosphere assays, which are also used to evaluate progenitor proliferation, is that it permits complete registering of individual cell divisions and behavior and a clear analysis of intrinsic mechanisms at work in choosing cell fate. Neurospheres, which are essentially a collection of cells arising from a progenitor proliferating in suspension, do not permit such analysis. However, the neurosphere culture is a useful assay to expand progenitors () using extrinsic factors. Neurospheres thus generated can then be differentiated on an adhesive substratum to generate neurons and glia.
An obvious drawback of this method of clonal analysis of isolated cortical progenitors is that dissociated progenitors neither encounter the permissive microenvironments (i.e., progenitor niches) found in vivo nor do they maintain critical features such as polarity and orientation they would have in the intact brain. This limitation might have major consequences considering that coordinated patterns of symmetric and asymmetric progenitor divisions in vivo rely on appropriate cell–cell contacts (Lu et al. 2001
) that are absent in isolated cultures. Cortical slice assays help overcome these limitations of isolated progenitor cultures.
Cerebral Cortical Slice Assays for Real-Time Analysis of Progenitor Development
Improvements in tissue culture methods and live-cell imaging in tissue slices have made possible the real-time tracking of individual progenitors and their progeny within tissue explants. Currently, this involves targeted expression of a fluorescent protein in progenitors followed by time-lapse confocal imaging of slice cultures. Depending on the objective of the experiment, single or multiple progenitors can be targeted in specific proliferative regions of both embryonic and postnatal cortices. The most common modes of fluorescent gene expression rely on electroporation (in utero and ex utero), viral transduction, and germ line genetic manipulation, each having unique advantages. For example, while viral transduction enables tracking of individual cells in isolation, electroporation and genetic manipulation allow targeting of cohorts of specific progenitor subpopulations.
Likely the most rapid method of progenitor transfection, DNA electroporation results in 1 or more regions of the cortex expressing a fluorescent transgene in multiple progenitors and their neuronal and glial progeny. In this method, concentrated (2–5 mg/mL) purified plasmid DNA containing the gene of interest is injected with a micropipette into the lateral ventricle of embryos, either removed from the uterus (ex vivo) (Gongidi et al. 2004
) or left intact in the embryonic sac (in utero) (Tabata and Nakajima 2001
). A low-voltage current is then passed through the cortex, and the DNA is incorporated into progenitors lining the lateral ventricles. The current's path can be oriented differently so that plasmid DNA will be taken up preferentially by progenitors that fall within the current's path. In this way, progenitors in the dorsal (Tabata and Nakajima 2001
), ventral, and hippocampal (Navarro-Quiroga et al. 2007
) anlages can be exclusively targeted. After ex utero electroporation, the brains are removed, sliced, and maintained in vitro. After sufficient time for transgene expression (24–48 h), long-term live-cell imaging can be performed on labeled progenitors in slices to analyze their patterns of differentiation (Xie et al. 2007
; Yoon et al. 2008
; Yokota et al. 2009
) using a confocal microscope equipped with live-cell incubation system. With in utero electroporation, the embryos are returned to the mother, where they develop for the desired number of additional days. The advantage of the ex utero approach is that it avoids the potential for embryonic death that, at times, occurs from the insults associated with in utero electroporation and allows for rapid analysis of large numbers of cortical progenitors. In contrast, in utero electroporation allows the cortex to develop intact until the effect of transgene expression in progenitors is ready to be analyzed.
In its simplest form, electroporation followed by live-cell imaging is a powerful method for observing the dynamics of progenitors as they differentiate, divide, and generate neurons. With the development of brighter, stable fluorescent markers, critical features of progenitors such as interkinetic nuclear movement, interprogenitor interactions, and pial end-feet dynamics can be followed in RGPs in both normal cortex and models of neurodevelopmental disorders (Xie et al. 2007
; Yokota et al. 2009
). Moreover, combining fluorescent labeling with genetic manipulation of progenitors, by coelectroporating mutated genes or short hairpin RNA (shRNA) constructs, can be an extremely efficient way to analyze the molecular mechanisms that regulate progenitor morphology, proliferation, and neurogenesis (LoTurco et al. 2009
; Loulier et al. 2009
). For example, the efficiency and reproducibility of electroporation make this technique amenable to rapid screening with shRNA libraries or shRNA screens targeting specific susceptibility genes for neurodevelopmental disorders. Further, by varying the timing of electroporation and using progenitor-type specific promoters, one can target and study radial progenitors and their progeny at distinct stages of development (Mizutani et al. 2007
; Anthony and Heintz 2008
). Postnatally, electroporation can be used to label and study progenitors and new neurons in the neurogenic niches (e.g., SVZ) of the neonatal or mature brain (Barnabe-Heider et al. 2008
) This approach can significantly facilitate our ability to study adult neurogenesis and the response of adult neural progenitors to injury and disease.
Viral transduction offers an alternative to electroporation. In this technique, high-titer retroviral particles expressing a gene of interest and a fluorescent marker are injected into the lateral ventricle 1–2 days prior to slice culture and imaging. The viral particles diffuse evenly through the ventricular space and infect single isolated progenitors, which can then be visualized and tracked as they divide and generate daughter neurons (Noctor et al. 2001
). Because viral vectors stably integrate into the genome, both progenitors and their progeny will express the transgene at relatively consistent levels. This is in contrast to electroporated cells where the plasmid DNA does not always integrate, and expression levels can vary widely among cells. However, when analysis of large cohorts of progenitors and rapid gene manipulation are required, electroporation-based methods are optimal.
Advanced Techniques to Study Progenitor Development in Intact Brains
In spite of many advantages, the isolated progenitor cell culture methods and the slice-based assays do not entirely preserve the neural progenitor cell niche. Neural stem cell niche in the developing brain, consisting of the extracellular matrix, inter-progenitor adhesive contacts, and the vasculature within the proliferative zone, can dynamically influence the appropriate unfolding of neural progenitor division and differentiation (Chenn and Walsh 2003
; Klezovitch et al. 2004
; Shen et al. 2004
; Rasin et al. 2007
; Loulier et al. 2009
; Stubbs et al. 2009
; Weimer et al. 2009
). As such, it will be vital to develop methods to observe and study neural progenitor development in live embryonic brains in utero. Multiphoton microscopy-based methods can be employed to evaluate neural progenitor development in the intact developing cerebral cortex in utero (see Supplementary Figure 1
and Supplementary Movie 1
). Embryonic mice expressing X
FP in neural progenitors can be used to image specific types of neural progenitors at different stages of cortical development in vivo. Although technical issues such as mechanical stability of embryos necessary for repeated long-term imaging of an identical area of the proliferative zone and the increasing thickness of cerebral wall during development can present obstacles for this type of imaging, the ability to follow neural progenitor differentiation and behavior in utero holds great promise for our understanding of how patterns of progenitor differentiation influence the formation of cerebral cortex.